METHOD FOR DETERMINING THE ALLELE FREQUENCY/MUTATION RATE, AND DIAGNOSTICS

Abstract

The present invention relates to a new method for determining the allele frequency and/or mutation rate in nucleic acids, in particular in tumor nucleic acids, in the context of a polymerase chain reaction (PCR), and to diagnostics for this purpose, wherein at least one reference nucleic acid (RN) and one mutation sequence with respect to the reference nucleic acid are used. This reference nucleic acid and mutation sequence allows polymerase chain reaction (PCR) methods to be validated, in particular on the basis of device parameters and sample preparation. Furthermore, the invention relates to an associated diagnosis and prognosis method, in particular for tumor diagnosis as part of a liquid biopsy.

Claims

1.-13. (canceled)

14. Method for validating a polymerase chain reaction (PCR) method by means of determining the allele frequency and/or mutation rate in nucleic acids, comprising the steps of: a.) providing at least one human reference nucleic acid and one human nucleic acid having one or more mutations relative to the reference nucleic acid in a DNA-free serum or plasma sample, wherein a particular allele frequency and/or mutation rate is predetermined, and optionally b.) providing a human reference nucleic acid from a.) in a DNA-free serum or plasma sample, and optionally c.) providing a DNA-free serum or plasma sample, wherein the particular allele frequency in a.) is detected, and where necessary is compared with b.) and/or c.).

15. Method for validating a PCR method by means of determining the allele frequency and/or mutation rate in nucleic acids according to claim 14, characterized in that the method is carried out for a plurality of predetermined allele frequencies from a.).

16. Method for validating a PCR method by means of determining the allele frequency and/or mutation rate in nucleic acids according to claim 15, characterized in that a validation and calibration curve is obtained.

17. Method for validating a PCR method for determining the allele frequency and/or mutation rate in nucleic acids according to claim 14, characterized in that the concentrations of reference nucleic acid and mutation nucleic acids in a.) and b.) are predetermined.

18. Method for validating a PCR method for determining the allele frequency and/or mutation rate in nucleic acids according to claim 14, characterized in that the mutation nucleic acids comprise at least one tumor marker.

19. Method for validating a PCR method for determining the allele frequency and/or mutation rate in nucleic acids according to claim 14, characterized in that a qPCR (real-time quantitative polymerase chain reaction), digital droplet PCR (ddPCR), or “next generation sequencing” (NGS) is carried out.

20. Method for determining the allele frequency and/or mutation rate of at least one sample nucleic acid by means of a PCR method, wherein calibration is carried out by means of a method according to claim 14.

21. Method for determining the allele frequency and/or mutation rate of at least one sample nucleic acid by means of a PCR method, wherein the sample nucleic acid is quantitatively determined.

22. Method for determining the allele frequency and/or mutation rate of at least one sample nucleic acid by means of a PCR method according to claim 20, characterized in that the sample nucleic acid of a patient is a cfDNA or ctDNA.

23. Method for the diagnosis or prognosis of a tumor disease, wherein a change in the allele frequency and/or mutation rate of a sample nucleic acid from a first sample and a second and/or further sample allows for early detection and detection, for the degree of severity to be assessed, and for progression to be assessed accompanied by treatment, wherein calibration is carried out by means of a method according to claim 14.

24. Method for the diagnosis or prognosis of a tumor disease according to claim 23, wherein the second or further sample is taken from a patient at a later point in time.

25. Kit containing a.) part having at least one human reference nucleic acid and one nucleic acid having one or more mutations relative to the reference sequence in a DNA-free serum or plasma sample, wherein a predetermined allele frequency is set, and optionally b.) part having a human reference nucleic acid in a DNA-free serum or plasma sample, and optionally c.) part having a DNA-free serum or plasma sample, for carrying out a method according to claim 14.

Description

EXAMPLE 1

[0044] Carrying out the method according to the invention by providing a kit:

1) Extraction of nucleic acids (DNA), in particular cfDNA, from the serum or plasma reference material according to the invention using commercial kits, such as the QIAamp ccfDNA/RNA kit (Qiagen®), the PME Free-Circulating DNA Extraction Kit (AnalytikJena®), or the MagMAX™ Cell-Free DNA Isolation Kit (ThermoFisher®)).
2) Quantification of the nucleic acids, in particular cfDNA in the eluate fluorometrically e.g. using Qubit® (ThermoFisher®) or spectrophotometrically using NanoDrop® or another spectrometer.
3) Optionally, a qualitative analysis of the nucleic acids, in particular cfDNA, can be carried out, such as a fragment length analysis using a bioanalyzer (Agilent®), fragment analyzer (Agilent®)), or pulsed-field gel electrophoresis.
4) The obtained nucleic acid, in particular cfDNA, is then supplied to a PCR.

[0045] At the same time, a corresponding serum or plasma sample from a patient can be prepared.

Output ddPCR:

[0046] Using the example of a QX200 ddPCR system from BioRad®:

[0047] By means of ddPCR, a defined volume of the nucleic acid to be tested is fractionated into thousands of individual reaction chambers. In this process, the DNA sequences to be tested are fractionated into these reaction chambers using Poisson distribution. Amplification of the nucleic acids takes place in the reaction chambers if the nucleic acid is present. The mutation sequence triggers a PCR reaction that can be differentiated in color from the reference sequence (wild type sequence).

[0048] By means of relevant software, the results can be evaluated and represented in a plot, in particular a 2D plot of amplitude:

[0049] The results detected in the FAM channel (blue fluorescence) (channel 1 amplitude) are plotted on the Y axis.

[0050] The results detected in the HEX/VIC channel (green fluorescence) (channel 2 amplitude) are plotted on the X axis.

[0051] Each plotted point represents a reaction chamber by a reaction having been carried out (FIG. 1a) with a reference sequence (wild type sequence) (Q4), a mutation sequence (Q1), with both sequences (Q2), or with no sequence (Q4), because no target nucleic acid was present, the obtained point clouds Q1 to Q4 being shown in a graph in FIG. 1b.

[0052] An evaluation takes place such that the raw data (plotted points) determined by the software provide numbers quantified in an absolute manner for the reference sequence and the mutation sequence.

Table 1: Example values for Q1 and Q4:

TABLE-US-00002 Well Data Well DyeName (s) Copies/20 μWell C11 FAM 477 C11 VIC 10632

[0053] The allele frequency can be determined from these values, e.g. manually or using software, i.e. the copy numbers for the reference sequence and the mutation sequence are calculated in accordance with the following formula for the allele frequency:

(CN.sub.Mut*100%)/(CN.sub.Mut+CN.sub.Wt)=VAF or MAF

CN.sub.Mut=copy numbers of mutation sequence
CN.sub.wt=copy numbers of reference sequence
VAF=variation in allele frequency or MAF=mutation in allele frequency.

[0054] In this way, the tubes of the kit (relevant standard sample with preset allele frequency, such as 0.0, 0.1, 1, and 5% in FIG. 2) undergo the method according to the invention, with the following information advantageously being obtained:

1) Qualitative: is the mutation detectable in the mutation sequence, yes/no?
2) Copy numbers (“CN”) quantified in an absolute manner for the reference sequence and mutation sequence,
3) Determining the allele frequency.

[0055] Examples are set out in FIG. 2.

[0056] A calibration curve can be compiled from the calculated values (FIG. 2, table) and the validation can be completed (FIG. 3).

[0057] Simple evaluation is also possible by means of NGS, since each copy corresponds to a “read,” and the reference sequence and mutation sequence can likewise be differentiated in a simple manner.

[0058] Patient or test-subject samples (supra) can be determined at the same time or different times on the basis of the calibration curve and a diagnosis can be made.